The neocortex plays a central role in many higher-order functions such as the interpretation of sensory information, comprehension of language and control of voluntary movements. Many of these processes are shaped by ascending cholinergic drive from neurons in the basal forebrain complex, principally nucleus basalis. Dysfunction of this pathway leads to deficits in many behaviors that involve neocortex and has been tied to clinical conditions such as depression, Parkinson's disease and Alzheimer's disease. Our long-term aim is to understand the cellular and network mechanisms by which ACh from nucleus basalis so profoundly influences cortical function. ACh acts at muscarinic and nicotinic ACh receptors (mAChRs and nAChRs), which are widely expressed in neocortex. Pyramidal neurons in neocortex express both of these receptor types, but the physiological functions of nAChRs in pyramidal neurons are unknown. In this proposal we describe the functions of nAChRs on pyramidal neurons. We directly assess how ACh, released by axons from nucleus basalis, affects pyramidal neurons in motor cortex, using a combination of techniques, including optogenetics, viral tools, genetically-modified mice, immuno-cytochemistry, cellular electrophysiology, two-photon microscopy and electron microscopy. We express channelrhodopsin-2 in cholinergic neurons in nucleus basalis and their axons in neocortex. In preliminary experiments, activation of cholinergic axons depolarized and promoted spiking of layer 5 pyramidal neurons via nAChRs, leading us to hypothesize that ACh facilitates the transfer of information through neocortical networks from ascending excitatory inputs, e.g. from thalamus, to target structures, e.g. motor circuits in the spinal cord. In this proposal we will determine whether the effects of ACh on pyramidal neurons support this hypothesis. We will investigate the mechanisms by which nAChRs affect spiking and the nAChR receptor subunits involved (specific aim 1), determine where in the activated nAChRs are located within the dendritic trees of pyramidal neurons (specific aim 2), and determine whether these postsynaptic nAChRs mediated the effects of ACh in pyramidal neurons in other layers and neocortical areas (specific aim3). Our results will reveal a new mechanism by which ACh modulates the excitability of pyramidal neurons. Our studies will also provide the first evidence, to my knowledge, that ACh has layer-specific effects. The resulting hypothesis has the potential to transform our understanding of the manner in which the cholinergic pathway from nucleus basalis changes network function in neocortex and may therefore have important implications for debilitating conditions such as Parkinson's disease and Alzheimer's disease. PUBLIC HEALTH RELEVANCE: The actions of the neuromodulator acetylcholine in neocortex play central roles many higher-order functions, including arousal, attention and learning and memory, and insufficient acetylcholine in neocortex has been tied to pathophysiological conditions such as Alzheimer's disease. He we study the locations of nicotinic acetylcholine receptors on layer 5 pyramidal neurons in the neocortex and the physiological effects of synaptically-released acetylcholine on these neurons. This study will advance our understanding of the mechanism of action of this essential neuromodulator in the healthy brain, which may also lead to insights into pathological conditions such as Alzheimer's disease.